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WO2018163163A1 - Compositions et méthodes pour le traitement de maladies inflammatoires - Google Patents

Compositions et méthodes pour le traitement de maladies inflammatoires Download PDF

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Publication number
WO2018163163A1
WO2018163163A1 PCT/IL2018/050248 IL2018050248W WO2018163163A1 WO 2018163163 A1 WO2018163163 A1 WO 2018163163A1 IL 2018050248 W IL2018050248 W IL 2018050248W WO 2018163163 A1 WO2018163163 A1 WO 2018163163A1
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WIPO (PCT)
Prior art keywords
composition
thca
fraction
subject
cells
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/IL2018/050248
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English (en)
Inventor
Hinanit KOLTAI
Yoram Kapulnik
Moran MAZUZ
Timna NAFTALI
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Mor Research Applications Ltd
Israel Ministry of Agriculture and Rural Development
Agricultural Research Organization of Israel Ministry of Agriculture
Original Assignee
Mor Research Applications Ltd
Israel Ministry of Agriculture and Rural Development
Agricultural Research Organization of Israel Ministry of Agriculture
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Application filed by Mor Research Applications Ltd, Israel Ministry of Agriculture and Rural Development, Agricultural Research Organization of Israel Ministry of Agriculture filed Critical Mor Research Applications Ltd
Priority to EP18764840.7A priority Critical patent/EP3592345A4/fr
Priority to CA3055271A priority patent/CA3055271A1/fr
Priority to US16/491,197 priority patent/US11833131B2/en
Priority to CN201880026618.1A priority patent/CN110799186A/zh
Priority to IL269158A priority patent/IL269158B2/en
Publication of WO2018163163A1 publication Critical patent/WO2018163163A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/658—Medicinal preparations containing organic active ingredients o-phenolic cannabinoids, e.g. cannabidiol, cannabigerolic acid, cannabichromene or tetrahydrocannabinol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/348—Cannabaceae
    • A61K36/3482—Cannabis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention in some embodiments thereof, relates to compositions and methods for the treatments of inflammatory diseases.
  • IBDs Inflammatory bowel diseases
  • CD Crohn's disease
  • UC ulcerative colitis
  • Epithelial cells in the gastrointestinal (GI) tract act as barriers against the intrusion of potentially deleterious luminal substances and microorganisms from the intestinal lumen, and play an important role in inflammatory responses. They express a variety of pro -inflammatory cytokines, which are upregulated in IBD patients [2]. Therapies aimed at down-regulating intestinal inflammation utilize both mediator- specific and nonspecific immune suppression, but with potentially considerable side effects [3].
  • C. sativa contains more than 60 terpenophenolic compounds termed phytocannabinoids (reviewed by [6]).
  • phytocannabinoids ⁇ 9- tetrahydrocannabinol (THC) and cannabidiol (CBD), which were discovered about 50 years ago, have been defined as the most active [7-9].
  • Cannabinoids have been previously shown to be immune modulators. They shift the balance of pro- and anti-inflammatory cytokines and act to suppress cell-mediated immunity in different physiological systems [10]. For example, ⁇ 9- tetrahydrocannabivarin (THCV) was demonstrated to inhibit nitrite production in macrophages and thereby to play an immunomodulatory role [11].
  • Cannabinoids derived from Cannabis sativa have been shown to exert their biological functions on the GI tract by mainly activating two types of G-protein-coupled cannabinoid receptors: cannabinoid type 1 (CB l) and cannabinoid type 2 (CB2) [4, 12]. Later, a third cannabinoid receptor, GPR55, was identified [13]. These receptors are part of the endocannabinoid system in the GI tract [13,14].
  • Cannabinoids have been shown to be effective in a mouse model of colitis [15].
  • HT29 human colonic epithelial cell line
  • cannabinoid receptor agonists and antagonists including the plant-derived THC, have been shown to inhibit tumor necrosis factor alpha (TNF-a)-induced interleukin-8 (IL-8) release [12]. This inhibition was antagonized by a CB2 receptor antagonist.
  • Cannabinoids have also been shown to promote wound healing in the GI tract via CB l receptor activation [4,16].
  • IBD patients we have recently reported clinical data from IBD patients. In a retrospective study we interviewed 30 CD patients who were licensed to use medical cannabis [17], while in a prospective trial we randomized 20 CD patients to receive either cannabis or placebo for their IBD [18]. Both revealed beneficial effects.
  • C. sativa extracts contain hundreds of different compounds. The activity of many synthetic or isolated cannabinoids and their receptor agonists or antagonists have been investigated and verified. However, there seems to be an advantage of the unrefined content of the flower versus an isolated compound in IBD.
  • cannabidiol CBD
  • Cannabis sativa extract with high content of cannabidiol CBD was shown in animal model of GI inflammation to attenuate injury and motility, once given after the inflammatory insult, further sustaining the rationale of combining CBD with other minor Cannabis constituents [19].
  • CBD cannabidiol
  • cannabis-derived botanical drug substances exerted significant anticonvulsant effects in three models of seizure and were of comparable efficacy with purified cannabidivarin (CBDV) [20].
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a liquid chromatography fraction of a cannabis extract comprising at least 75 % tetrahydrocannabinolic acid (THCA), wherein the fraction comprises cannabis derived active ingredients other than the THCA, thereby treating the inflammatory disease in the subject.
  • THCA tetrahydrocannabinolic acid
  • a therapeutically effective amount of a liquid chromatography fraction of a cannabis extract comprising at least 75 % tetrahydrocannabinolic acid (THCA), wherein the fraction comprises cannabis derived active ingredients other than the THCA, for use in treating an inflammatory disease in a subject in need thereof.
  • THCA tetrahydrocannabinolic acid
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a composition of matter comprising a therapeutically effective amount of liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, wherein the active ingredients comprise THCA, thereby treating the inflammatory disease in the subject.
  • a therapeutically effective amount of liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, wherein the active ingredients comprise THCA, for use in treating an inflammatory disease in a subject in need thereof.
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising tetrahydrocannabinolic acid (THCA), wherein the THCA constitutes at least 30 % of the active ingredients in the composition, thereby treating the inflammatory disease in the subject.
  • THCA tetrahydrocannabinolic acid
  • THCA tetrahydrocannabinolic acid
  • composition comprising liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, the active ingredients comprising THCA.
  • composition comprising liquid chromatography-purified cannabis fraction obtainable by subjecting the cannabis extract to liquid chromatography and collecting fractions detectable by a detector operated at 220 nm.
  • compositions comprising liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, the composition being characterized by: (i) having a cytotoxic activity on cancer cells; (ii) reducing the level of pro-inflammatory cytokine secretion by cells; and/or (iii) reducing the level of MMP9 and COX2 expression in cells.
  • composition comprising THCA and CBD, wherein the composition is devoid of at least one of cannabichromene (CBC), cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), cannabigerol (CBG) and/or cannabinol (CBN).
  • CBC cannabichromene
  • CBD cannabigerolic acid
  • CBDA cannabidiolic acid
  • CBD cannabigerol
  • CBN cannabinol
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of some embodiments of the invention, thereby treating the inflammatory disease in the subject.
  • composition of some embodiments of the invention for use in treating an inflammatory disease in a subject in need thereof.
  • a method of generating an anti-inflammatory composition comprising: (i) adding a polar solvent to a dry Cannabis inflorescence so as to obtain a crude extract; (ii) filtering the crude extract so as to obtain a filtered extract; (iii) fractionating the filtered extract on a high pressure liquid chromatography (HPLC); (iv) collecting at least one fraction comprising active ingredients detectable by a detector operated at 220 nm.
  • HPLC high pressure liquid chromatography
  • an anti-inflammatory composition obtainable by the method of some embodiments of the invention.
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of some embodiments of the invention, thereby treating the inflammatory disease in the subject.
  • composition of some embodiments of the invention for use in treating an inflammatory disease in a subject in need thereof.
  • a method of determining an anti-inflammatory activity of the composition of any one of some embodiments of the invention comprising ex- vivo contacting an inflamed tissue of a subject with the composition, wherein an increased anti-inflammatory response of the inflamed tissue above a predetermined threshold is indicative of the anti-inflammatory activity of the composition.
  • the composition comprises THCA.
  • the composition comprises at least 75 % THCA.
  • the composition comprises at least one of D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), ⁇ 9- tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the composition comprises D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and cannabigerol (CBG).
  • the composition comprises at least one of the components listed in Table 6.
  • the composition is characterized by: (i) having a cytotoxic activity on cancer cells (ii) reducing the level of pro -inflammatory cytokine secretion by cells; and/or (ii) reducing the level of MMP9 and COX2 expression in cells.
  • the composition comprises CBD.
  • the method further comprises administering to the subject a therapeutically effective amount of cannabidiol (CBD).
  • CBD cannabidiol
  • the fraction, THCA or composition for use further comprises the use of a therapeutically effective amount of cannabidiol (CBD).
  • CBD cannabidiol
  • the method further comprises administering to the subject an agonist of CB 1 receptor, CB2 receptor and/or GPR55.
  • the fraction, THCA or composition for use further comprises the use of an agonist of CB 1 receptor, CB2 receptor and/or GPR55.
  • the THCA comprises a synthetic THCA or analog thereof having an anti-inflammatory activity.
  • the THCA comprises a liquid chromatography fraction of a cannabis extract, the fraction comprising at least 75 % tetrahydrocannabinolic acid (THCA), wherein the fraction comprises cannabis derived active ingredients other than the THCA.
  • THCA tetrahydrocannabinolic acid
  • the cannabis extract comprises about 80- 95 % THCA.
  • the fraction or fractions comprise at least one of D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), ⁇ 9- tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the fraction or fractions comprises at least two of the D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the fraction or fractions comprises components as listed in Table 6.
  • the CBD comprises a synthetic CBD or analog thereof having an anti-inflammatory activity.
  • the inflammatory disease is an inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the IBD is selected from the group consisting of ulcerative colitis and Crohn's disease.
  • the subject is a human subject.
  • the liquid chromatography comprises high pressure liquid chromatography (HPLC).
  • the liquid chromatography is performed on a reverse stationary phase.
  • the liquid chromatography is performed using a mobile phase comprising from 10 to 30 % acidic aqueous solution and from 90 to 70 % alcohol.
  • the HPLC comprises a stationary phase comprising RP-18 end capped column, and a mobile phase of 15 % solvent A (0.1 % acetic acid in water) and 85 % solvent B (methanol) at a flow rate of 1.5 mL/min for 28-35 minutes.
  • the conditions for the HPLC comprise an UltiMate 3000 HPLC system coupled with WPS-3000(T) Autosampler, HPG-3400 pump, and DAD-300 detector, a Purospher RP-18 end capped column a mobile phase of 15% solvent A (0.1% acetic acid in water) and 85% solvent B (methanol) at a flow rate of 1.5 mL/min for 28-35 minutes.
  • the detector is a diode array detector. According to some embodiments of the invention, the at least one fraction comprises
  • the at least one fraction comprises at least 75% THCA.
  • the at least one fraction comprises at least one of D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the at least one fraction comprises components as listed in Table 6.
  • the HPLC comprises a stationary phase comprising RP-18 end capped column, and a mobile phase of 15 % solvent A (0.1 % acetic acid in water) and 85 % solvent B (methanol) at a flow rate of 1.5 mL/min for 28-35 minutes.
  • the conditions for the HPLC comprise an UltiMate 3000 HPLC system coupled with WPS-3000(T) Autosampler, HPG-3400 pump, and DAD-300 detector, a Purospher RP-18 end capped column a mobile phase of 15 % solvent A (0.1 % acetic acid in water) and 85 % solvent B (methanol) at a flow rate of 1.5 mL/min for 28-35 minutes.
  • the detector is a diode array detector.
  • the inflamed tissue is a gastrointestinal tissue biopsy.
  • the anti-inflammatory activity of the composition comprises an upregulation in secretion of an anti-inflammatory factor and/or reduction in secretion of a pro-inflammatory factor.
  • the anti-inflammatory activity of the composition comprises reduction in expression of a gene associated with the inflammation.
  • FIGs. 1A-D depict ( Figure 1A) Determination of HCT116 cell viability using Alamar Blue fluorescence (Resazurin assay) as a function of cell number.
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a and C. sativa ethanolic extracts of fresh (C2F, 0.2 mg/mL) or baked (C2B, 0.2 mg/mL) cannabis flowers, 20 ⁇ dexamethasone for 16hours.
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a and C2F, C2B for 16hours. Following, the cells were incubated with Alamar Blue for 4hours.
  • FIGs. 1E-F depict ( Figure IE) Anti-inflammatory activity at different concentrations of
  • Cannabis sativa ethanolic fresh flower extracts (C2F; 114-207 ⁇ g/mL), baked flower extracts (C2B; 114-207 ⁇ g/mL), F7 from fresh flower extracts (an HPLC fraction of C2F at concentrations of 114-207 ⁇ g/mL), and Dex at 200 and 400 ⁇ on HCT 116 cells measured as level of IL-8 (ng/mL).
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37 °C in a humidified 5 % C02-95 % air atmosphere. Cells were treated with 300 ng/mL TNF-a and 50 ⁇ ⁇ of C.
  • sativa ethanol extract of C2F or fractions for 4 hours Non- treated are the cells without TNF-a and treatments. Levels of IL-8 were measured from the supernatant using a commercial kit. Values (ng/mL) were calculated relative to a TNF-a-treated control.
  • Figure IF Determination of HCT116 cell viability using Alamar Blue fluorescence (resazurin assay) as a function of live cell number. Cells were seeded and treated as described in ( Figure IE). Next, the cells were incubated with Alamar Blue for 2 hours. Relative fluorescence at the excitation/emission of 544/590 nm was measured.
  • FIGs. 1G-H depict ( Figure 1G) Anti-inflammatory activity at different concentration of C. sativa ethanolic fresh flower extracts (C2F; 114 ⁇ g/mL to 207 ⁇ g/mL), baked flower extracts (C2B; 114 ⁇ g/mL to 207 ⁇ g/mL), F7 from fresh flower extracts (an HPLC fraction of C2F at concentrations of 114 ⁇ g/mL to 207 ⁇ g/mL) and dexamethasone (Dex) at 200 and 400 ⁇ on HT 29 cells measured as level of IL-8 (ng/ml).
  • C2F C. sativa ethanolic fresh flower extracts
  • C2B baked flower extracts
  • Dex dexamethasone
  • HT 29 were seeded (50,000 per well) in triplicate in 500 ⁇ L ⁇ growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere.
  • Cells were treated with 300 ng/mL TNF-a and 50 ⁇ ⁇ of C. sativa ethanol extract of C2F or fractions for 4 hours.
  • Non-treated are the cells without TNF-a and treatments.
  • Levels of IL- 8 were measured from the supernatant using a commercial kit. Values (ng/mL) were calculated relative to a TNF-a-treated control.
  • FIG. 1H Determination of HT29 cell viability using Alamar Blue fluorescence (Resazurin assay) as a function of live cell number.
  • FIGs. II- J depict ( Figure II) Anti-inflammatory activity at different concentration of C.
  • C2F sativa ethanolic fresh flower extracts
  • C2B baked flower extracts
  • F7 from fresh flower extracts an HPLC fraction of C2F at concentrations of 114 ⁇ g/mL to 207 ⁇ g/mL
  • Dex dexamethasone
  • CaC02 were seeded (50,000 per well) in triplicate in 500 growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere.
  • FIGs. 2A-B depicts HPLC chromatograms of C. sativa ethanolic extracts. Chromatogram of fresh cannabis extract ( Figure 2B, C2F, 0.1 mg/mL) and baked (i.e., fresh flowers which were baked at 150 °C for 3 hours) cannabis extract ( Figure 2A, C2B, 0.1 mg/mL) obtained from isocratic elution with a mixture of 15 % water containing 0.1 % acetic acid (solvent A) and 85 % MeOH (solvent B) for a total run time of 35minutes at 220 nm. The samples were injected at a concentration of 0.58 mg/mL in a volume of 20 ⁇ ⁇ for C2B and 0.33 mg/mL in a volume of 20 ⁇ ⁇ for C2F.
  • FIGs. 3A-D depict ( Figure 3 A) HPLC profile of fractions of C. sativa ethanolic extract.
  • HPLC profile was obtained from isocratic elution with a mixture of 15 % water containing 0.1 % acetic acid (solvent A) and 85 % MeOH (solvent B) for a total run time of 40minutes at 220 nm.
  • the sample was injected at a concentration of 0.1 mg crude dried extract/mL in a volume of 50 ⁇ ⁇ per cycle. Fractions were collected every 2minutes.
  • F1-F9 represent the nine fractions into which the peaks were divided.
  • FIG. 3B Anti-inflammatory activity of fractions F1-F9 pooled together, F1-F9 pooled together without F7, Fl - F9 separately from fresh flowers (C2F, 0.9 mg/mL) of C. sativa ethanolic extracts measured as level of IL-8 (ng/mL) on HCT116 cells.
  • Figure 3C Antiinflammatory activity of fractions F1-F9 pooled together without F7, F1-F9 pooled together with F7, F7 from fresh flowers (C2F) of C.
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a and 50 ⁇ ⁇ of C. sativa ethanol extract of C2F or fractions for 16hours. Non-treated are the cells without TNF- ⁇ and treatments. Levels of IL-8 were measured from the supernatant using a commercial kit.
  • HT29 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere.
  • Cells were treated with 50 ng/mL TNF-a and 50 ⁇ ⁇ C. sativa ethanol extracts of C2F, C2B, CBD or dexamethasone.
  • FIGs. 3E-F depict ( Figure 3E) Anti-inflammatory activity of C. sativa ethanolic extracts (C2F; 163 ⁇ g/mL), fractions F1-F9 pooled together (HPLC fractions of C2F at concentrations of 163 ⁇ g/mL), Fl-F9-excluding F7 (HPLC fractions of C2F at concentrations of 163 ⁇ g/mL), F1-F9 (each an HPLC fraction of C2F at concentrations of 163 ⁇ g/mL) on HCT 116 cells measured as level of IL-8 (ng/mL).
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37_°C in a humidified 5 % C02-95 % air atmosphere.
  • Cells were treated with 300 ng/mL TNF-a and 50 ⁇ ⁇ of C. sativa ethanol extract of C2F or fractions for 4 hours.
  • Non-treated are the cells without TNF-a and treatments.
  • Levels of IL-8 were measured from the supernatant using a commercial kit. Values (ng/mL) were calculated relative to a TNF-a- treated control.
  • FIG. 3F Determination of HCT 116 cell viability using Alamar Blue fluorescence (resazurin assay) as a function of live cell number.
  • FIGs. 3G-H depict ( Figure 3G) Anti-inflammatory activity of C. sativa ethanolic extracts (C2F; 163 ⁇ g/mL), F7 at three different concentrations (an HPLC fraction of C2F at concentrations of 142, 163, and 190 ⁇ g/mL), fractions F1-F9- excluding F7 (Fl-F9-exc F7) at two concentrations (HPLC fractions of C2F at concentrations of 163 and 190 ⁇ g/mL), combination of each concentration of Fl-F9-excluding F7 along with each concentration of F7 on HCT 116 cells measured as level of IL-8 (ng/mL).
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ L ⁇ growing media and incubated for 24 hours at 37_°C in a humidified 5 % C02-95 % air atmosphere.
  • Cells were treated with 300 ng/mL TNF-a and 50 ⁇ ⁇ of C. sativa ethanol extract of C2F or fractions for 4 hours.
  • Non-treated are the cells without TNF-a and treatments.
  • Levels of IL- 8 were measured from the supernatant using a commercial kit. Values (ng/mL) were calculated relative to a TNF-a- treated control.
  • FIGs. 3I-J depict ( Figure 31) Anti-inflammatory activity of cannabidiol (CBD) at different concentrations (16 ⁇ g/mL to 252 ⁇ g/mL) and dexamethasone (Dex; 200 and 400 uM), measured as level of IL-8 on HCT116, HT29 or CaC02 cells.
  • CBD cannabidiol
  • Dex dexamethasone
  • HCT116, HT29 or CaC02 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere.
  • FIG. 4 depicts a HPLC profile of C2F, F7 and THCA. Chromatograms of THCA standard at 40 ppm (marked in blue), whole C. sativa extract at 0.1 mg/mL (marked in green) and F7 at 0.04 mg/mL (marked in red). All samples were injected in a volume of 20 ⁇ ⁇ and were obtained from isocratic elution with a mixture of 15 % water containing 0.1 % acetic acid (solvent A) and 85 % MeOH (solvent B) for a total run time of 40minutes at 220 nm.
  • solvent A 0.1 % acetic acid
  • solvent B MeOH
  • FIG. 5 depicts the anti-inflammatory activity of F7 (0.07 mg/mL) from C. sativa fresh flowers ethanol extracts diluted accordingly to the concentration of 0.2 mM THCA, 0.2 mM THCA and 20 ⁇ dexamethasone, measured as level of IL-8 on HCT116 cells.
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a and F7, THCA, dexamethasone for 16hours. Levels of IL-8 were measured from the supernatant using a commercial kit.
  • FIG. 6 depicts the anti-inflammatory activity of pure CBD or 20 ⁇ dexamethasone, measured as level of IL-8 levels (ng/mL) on HCT116 cells.
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere.
  • Cells were treated with 50 ng/mL TNF-a and pure CBD at the different concentrations (2.5, 5, 7.5, 10 and 25 ⁇ ), or 20 ⁇ dexamethasone for 16hours.
  • Non-treated are the cells without TNF-a and treatments.
  • Levels of IL-8 were measured from the supernatant using a commercial kit.
  • FIG. 7 depicts Anti-inflammatory activity of C.
  • sativa F7 at three different concentrations an HPLC fraction of C2F at concentrations of 190 ⁇ g/mL), fractions Fl-F9-excluding F7 (F1-F9- exc F7; HPLC fractions of C2F at concentrations of 163 ⁇ g/mL), combination of Fl-F9-excluding F7 along with F7 measured as level of IL-8 on HCT116 cells, with and without antagonists to CB 1, CB2, and GPR55 receptors (antagonists at a concentration of 20 ⁇ , CB 1, CB 1 receptor antagonist rimonabant; CB2, CB2 receptor antagonist SR144528; GPR55, GPR55 antagonist CID16020046).
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37_°C in a humidified 5 % C02-95 % air atmosphere.
  • Cells were treated with 300 ng/mL TNF-a and 50 ⁇ ⁇ of C. sativa ethanol extract of C2F fractions for 4 hours.
  • Treatments with F7, F1-F9, and combination of fractions without antagonists served as a positive control (Con).
  • Non-treated are the cells without TNF-a and treatments.
  • Levels of IL-8 were measured from the supernatant using a commercial kit. Values (ng/mL) were calculated relative to control.
  • FIG. 8 depicts the anti-inflammatory activity of C. sativa fresh flowers ethanol extracts (C2F, 0.2 mg/ml) and F7 (0.08 mg/ml) measured as level of IL-8 and IL-6 from biopsies of Intestine Bowel Disease (IBD) patients.
  • Levels of IL-8 and IL-6 were measured from the supernatant using a commercial kit. Error bars indicate + SEM. *, **, *** indicates data statistically significantly different in comparison with the control (Non- treated tissues) at /? ⁇ 0.05, p ⁇ 0.001, p ⁇ 0.0001 respectively.
  • Levels with different letters are significantly different from all combinations of pairs by turkey HSD.
  • FIGs. 9A-B depicts COX2 and MMP9 gene expression.
  • Figure 9A HCT1 16 cell line. Cells were seeded (1,500,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a overnight and then treated with C. sativa C2F or F7 added 5hours before RNA extraction.
  • Figure 9B Biopsies. C2F and F7 were added overnight to four UC patients and one CD patient biopsies at concentrations of 0.2 mg/mL and 0.07 mg/mL, respectively.
  • the experiment was performed in three biological replicates, with three technical repeats for each (n 3).
  • FIG. 10 depicts anti-inflammatory (ELISA) assay on HT29 cells treated with different concentrations of fraction 7 of C.
  • sativa fresh flower extract measured as level of IL-8 on HT29 cells.
  • F7 from C. sativa fresh flowers ethanol extracts was diluted to 80, 60, 40, 30, 20 15, 10 and 5 ⁇ g/ml.
  • HT29 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a and F7 for 16 hours.
  • FIG. 11 depicts anti-inflammatory (ELISA) assay on HT29 cells treated with different concentrations of CBD, measured as level of IL-8 on HT29 cells.
  • CBD dissolved in methanol-at concentration of -5 mM was diluted to 80, 60, 40, 30, 20 15, 10 and 5 uM/ml.
  • HT29 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a and CBD for 16 hours. Levels of IL-8 were measured from the supernatant using a commercial kit.
  • FIG. 12 depicts anti-inflammatory (ELISA) assay on HT29 cells treated with different concentrations of THC, measured as level of IL-8 on HT29 cells.
  • THC dissolved in methanol-at concentration of -5 mM was diluted to 75, 50, 25 and 12.5 uM/ml.
  • HT29 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a and THC for 16hours. Levels of IL-8 were measured from the supernatant using a commercial kit.
  • FIGs. 13A-C depict EC50 dose of fractions on HCT116 cells.
  • Figure 13A Fraction 7 (F7),
  • Figure 13B CBD and
  • Figure 13C THC.
  • HCT116 cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 50 ng/mL TNF-a and F7, CBD or THC for 16 hours. Levels of IL-8 were measured from the supernatant using a commercial kit.
  • FIG. 14 depicts EC50 of F7 with and without CBD at its EC50 dose on HT29 cells.
  • Dose- effect curves of F7 with and without CBD on IL-8 levels of HT29 colon cancer cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 300 ng/mL TNF-a and F7 (at EC50 concentration) with and without CBD at different concentrations for 4 hours. Levels of IL-8 were measured from the supernatant using a commercial kit. Values (ng/mL) were calculated relative to a TNF-a treated control.
  • Non-treated are the cells without TNF-a and treatments.
  • data points were connected by non-linear regression lines of the sigmoidal dose-response relation.
  • GraphPad Prism was employed to produce dose-response curves.
  • FIG. 15 depicts EC50 of CBD with and without F7 at its EC50 dose on HT29 cells.
  • Dose- effect curves of CBD with and without F7 on IL-8 levels of HT29 colon cancer cells were seeded (50,000 per well) in triplicate in 500 ⁇ ⁇ growing media and incubated for 24 hours at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. Cells were treated with 300 ng/mL TNF-a and CBD (at EC50 concentration) with and without F7 at different concentrations for 4 hours. Levels of IL-8 were measured from the supernatant using a commercial kit. Values (ng/mL) were calculated relative to a TNF-a treated control.
  • Non-treated are the cells without TNF-a and treatments.
  • data points were connected by non-linear regression lines of the sigmoidal dose-response relation.
  • GraphPad Prism was employed to produce dose-response curves.
  • the present invention in some embodiments thereof, relates to compositions and methods for treating inflammatory diseases.
  • IBDs Inflammatory bowel diseases
  • CD Crohn's disease
  • UC ulcerative colitis
  • C. sativa inflorescence extracts were tested on epithelial cells, colon cells and colon tissues. It was shown that the anti-inflammatory activity of cannabis flower extracts derives from A9-tetrahydrocannabinolic acid (THCA).
  • THCA A9-tetrahydrocannabinolic acid
  • C. sativa polar extracts had a significant anti-inflammatory activity (see Example 1 of the Examples section which follows). This activity was also observed for fraction F7 which comprises mainly of THCA (see Examples 3, 5 and 12 of the Examples section which follows). However, C. sativa whole extract has an increased activity over THCA only (see Example 6 of the Examples section which follows).
  • a method of generating an anti-inflammatory composition comprising: (i) adding a polar solvent to a dry Cannabis inflorescence so as to obtain a crude extract; (ii) filtering the ethanol crude extract so as to obtain a filtered extract; (iii) fractionating the filtered extract on a high pressure liquid chromatography (HPLC); (iv) collecting at least one fraction comprising active ingredients detectable by a detector operated at 220 nm.
  • HPLC high pressure liquid chromatography
  • Cannabis is a genus of flowering plants in the family Cannabaceae that includes three different species, Cannabis sativa, Cannabis indica and Cannabis ruderalis.
  • the term Cannabis encompasses wild type Cannabis and also variants thereof, including cannabis chemovars which naturally contain different amounts of the individual cannabinoids. For example, some Cannabis strains have been selectively bred to produce high or low levels of THC and other cannabinoids. Accordingly, Cannabis cultivars that are rich in THCA can be used in accordance with the present teachings.
  • the Cannabis plant is a wild-type plant.
  • the Cannabis plant is transgenic.
  • the Cannabis plant is genomically edited.
  • the Cannabis plant is Cannabis sativa (C. sativa).
  • the extract may be derived from a cultivated Cannabis plant (i.e. not grown in their natural habitat) or may be derived from Cannabis plants which grow in the wild.
  • the tissue of the Cannabis plant from which the extract is typically obtained is the inflorescence. Accordingly, the extract may be obtained from the complete flower head of a plant including stems, stalks, bracts, and flowers. However, it will be appreciated that a cannabis extract of the invention may be obtained from only part of the inflorescence, such as from the bracts and/or flowers.
  • the extract is obtained from a fresh plant (i.e. a plant not heated prior to the extraction process).
  • Fresh plants include plants taken immediately following harvesting (e.g., up to an hour or several hours) for extraction as well as plants frozen immediately after harvesting (e.g. at about -70 °C to -90 °C, e.g. at -80 °C, for any required length of time) prior to extraction.
  • the extract is obtained from fresh inflorescence.
  • the extract is obtained from a frozen inflorescence (e.g. frozen immediately after harvesting at about -70 °C to -90 °C, e.g. at -80 °C, for any required length of time).
  • the extract may be obtained from a cryopreserved inflorescence, or from an inflorescence frozen in liquid nitrogen or in dry ice.
  • the extract is obtained from an inflorescence which has not been subjected to heating (such as heating at e.g. at 120 °C to 180 °C, e.g. at 150 °C, for any length of time, such as for 1-5 hours).
  • heating such as heating at e.g. at 120 °C to 180 °C, e.g. at 150 °C, for any length of time, such as for 1-5 hours.
  • the extract is obtained from dry Cannabis inflorescence. Drying the inflorescence may be carried out using any method known in the art, such as by pulverizing with liquid nitrogen or with dry-ice/alcohol mixture.
  • the polar solvent comprises a polar, protic solvent (e.g., ethanol or methanol). In some embodiments, the polar solvent comprises a polar, aprotic solvent (e.g., acetone).
  • Polar solvents suitable for use with the present invention include, but are not limited to, ethanol, methanol, n-propanol, iso-propanol, a butanol, a pentanol, acetone, methylethylketone, ethylacetate, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, water, and combinations thereof.
  • the polar solvent is ethanol (e.g. absolute ethanol, i.e. above
  • the concentration or amount of a polar solvent used to dry Cannabis inflorescence can be varied.
  • the ratio of a dry Cannabis inflorescence to a polar solvent is the amount of a polar solvent sufficient to extract about 70 % or more, about 75 % or more, about 85 % or more, about 90 % or more, about 95 % or more, about 97 % or more, or about 99 % or more of a composition having anti-inflammatory activity.
  • the ratio of polar solvent to dry Cannabis inflorescence is about 1:2 to about 1:20 (w/v), e.g. about 1:4 to about 1: 10 (w/v).
  • the extract is an ethanol extract.
  • absolute ethanol is added to the dry inflorescence at a sample-to- absolute ethanol ratio of 1:4 (w/v).
  • the dry Cannabis inflorescence is contacted with a polar solvent (e.g. ethanol) for about 15 minutes or more, about 30 minutes or more, about 1 hour or more, about 2 hours or more, or about 5 hours or more.
  • a polar solvent e.g. ethanol
  • the dry Cannabis inflorescence is contacted with a polar solvent at temperature of about 15 °C to about 35 °C, or about 20 °C to about 25 °C.
  • the dry Cannabis inflorescence is contacted with a polar solvent (e.g. ethanol) while being constantly mixed e.g. on a shaker.
  • a polar solvent e.g. ethanol
  • the process of the present invention comprises isolating a liquid extract (i.e. filtered extract) from the mixture (i.e. crude extract) comprising the liquid extract and solids.
  • a liquid extract i.e. filtered extract
  • Suitable means for isolating the liquid extract (i.e. filtered extract) include those known in the art of organic synthesis and include, but are not limited to, gravity filtration, suction and/or vacuum filtration, centrifuging, setting and decanting, and the like.
  • the isolating comprises filtering a liquid extract through a porous membrane, syringe, sponge, zeolite, paper, or the like having a pore size of about 1-5 ⁇ , about 0.5-5 ⁇ , about 0.1-5 ⁇ , about 1-2 ⁇ , about 0.5-2 ⁇ , about 0.1-2 ⁇ , about 0.5-1 ⁇ , about 0.1-1 ⁇ , about 0.25-0.45 ⁇ , or about 0.1-0.5 ⁇ (e.g. about 2 ⁇ , about 1 ⁇ , about 0.45 ⁇ , or about 0.25 ⁇ ).
  • the crude extract is filtered through a 0.45- ⁇ syringe filter such as that commercially available from Merck, Darmstadt, Germany.
  • the present inventors contemplate drying (i.e. removal of the polar solvent) and/or freezing the filtered extract following generation thereof.
  • the method for drying the filtered extract is not particularly limited, and can include solvent evaporation at a reduced pressure (e.g., sub- atmospheric pressure) and/or an elevated temperature (e.g., above about 25 °C).
  • a reduced pressure e.g., sub- atmospheric pressure
  • an elevated temperature e.g., above about 25 °C.
  • processes such as co-evaporation, lyophilization, and the like can be used to completely remove the polar solvent from a liquid fraction to form a dry powder, dry pellet, dry granulate, paste, and the like.
  • the polar solvent is evaporated with a vacuum evaporator.
  • the present inventors further contemplate additional purification steps so as to further purify active agents from the extract.
  • the present inventors further propose fractionating the filtered extract.
  • Fractionating can be performed by processes such as, but not limited to: column chromatography, preparative high performance liquid chromatography ("HPLC"), reduced pressure distillation, and combinations thereof. According to a specific embodiment, fractionating is performed by HPLC.
  • HPLC preparative high performance liquid chromatography
  • fractionating comprises resuspending the filtered extract in a polar solvent (such as methanol, as discussed above), applying the polar extract to a separation column, and isolating the Cannabis extract having anti-inflammatory activity by column chromatography.
  • a polar solvent such as methanol, as discussed above
  • An eluting solvent is applied to the separation column with the polar extract to elute fractions from the polar extract.
  • Suitable eluting solvents for use include, but are not limited to, methanol, ethanol, propanol, acetone, acetic acid, carbon dioxide, methylethyl ketone, acetonitrile, butyronitrile, carbon dioxide, ethyl acetate, tetrahydrofuran, di-iso-propylether, ammonia, triethylamine, N,N-dimethylformamide, ⁇ , ⁇ -dimethylacetamide, and the like, and combinations thereof.
  • liquid chromatography comprises high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • liquid chromatography is performed on a reverse stationary phase.
  • liquid chromatography is performed using a mobile phase comprising from 10 to 30 % acidic aqueous solution and from 90 to 70 % alcohol.
  • an eluting solvent comprises 15 % solvent A (0.1 % acetic acid in water) and 85 % solvent B (methanol).
  • fraction separation may be carried out on a HPLC comprising a stationary phase comprising RP-18 end capped column (such as a 250 mm x 4.6 mm available from e.g. Merck KGaA, Darmstadt, Germany) with a guard column (e.g. 4 mm x 4 mm), and a mobile phase of 15 % solvent A (0.1 % acetic acid in water) and 85 % solvent B (methanol) at a flow rate of 1.5 mL/min for 28-35 minutes.
  • RP-18 end capped column such as a 250 mm x 4.6 mm available from e.g. Merck KGaA, Darmstadt, Germany
  • guard column e.g. 4 mm x 4 mm
  • mobile phase 15 % solvent A (0.1 % acetic acid in water) and 85 % solvent B (methanol) at a flow rate of 1.5 mL/min for 28-35 minutes.
  • fractions comprising components (active ingredients) are detectable by a detector operated at 220 nm are collected.
  • the detector is a diode array detector.
  • the detector is a DAD-300 detector.
  • the conditions for HPLC include, for example, an
  • a Purospher RP-18 end capped column such as a 250 mm x 4.6 mm available e.g. from Merck KGaA, Darmstadt, Germany
  • guard column e.g. 4 mm x 4 mm
  • solvent A 0.1 % acetic acid in water
  • solvent B methanol
  • the extracts and/or fractions obtained may be tested for anti-inflammatory activity and/or for cytotoxic activity.
  • any in-vivo, in-vitro or ex-vivo assay known in the art for testing anti-inflammatory activity may be used.
  • in-vitro and ex-vivo assays may be used which analyze the effect of the extracts and/or fractions on cell derived factors such as IL-8, IL-6, IFN-gamma, leukotriene B4, nitric oxide, prostaglandins, TNF-alpha and IL-1.
  • common ELISA assays may be used.
  • in-vitro or ex-vivo assays may be used which analyze the effect of extracts and/or fractions on expression of genes associated with inflammation in cells. Such genes include, but are not limited to, COX2 expression (reviewed by [24]) and MMP9 (reviewed by [25]).
  • any in-vivo, in- vitro or ex-vivo assay known in the art for testing cytotoxic activity may be used.
  • cell viability assay on cancer cells e.g. colon cancer cells
  • the extracts and/or fractions of the present invention can also be characterized by analytical methods such as, but not limited to, spectroscopic methods such as, but not limited to, ultraviolet- visible spectroscopy ("UV-Vis”), infrared spectroscopy (“IR”), and the like; mass- spectrometry (“MS”) methods such as, but not limited to, time-of-flight MS; quadrupole MS; electrospray MS, Fourier-transform MS, Matrix- Assisted Laser Desorption/Ionization (“MALDI”), and the like; chromatographic methods such as, but not limited to, gas-chromatography (“GC”), liquid chromatograph (“LC”), high-performance liquid chromatography (“HPLC”), and the like; and combinations thereof (e.g., GC/MS, LC/MS, HPLC/UV-Vis, and the like), and other analytical methods known to persons of ordinary skill in the art.
  • analytical methods such as, but not limited to, spectroscopic methods such as, but not limited to, ultraviolet
  • the extracts and/or fractions obtained by the methods of some embodiments of the invention are kept frozen e.g. in a freezer, until further use (e.g. at about -20 °C to -90 °C, at about -70 °C to -90 °C, e.g. at -80 °C), for any required length of time.
  • the extracts and/or fractions obtained by the methods of some embodiments of the invention are immediately used (e.g. within a few minutes e.g., up to 30 minutes).
  • extracts and/or fractions obtained by the methods of some embodiments of the invention may be used separately.
  • different extracts e.g. from different plants or from separate extraction procedures
  • different fractions from the same extract, from different extracts, from different plants and/or from separate extraction procedures
  • pooled refers to collected from the liquid chromatography (e.g.
  • HPLC either as a single fraction or a plurality of fractions.
  • fractions may be obtained at the following retention times when the following conditions are used: HPLC comprise an UltiMate 3000 HPLC system coupled with WPS-3000(T) Autosampler, HPG-3400 pump, and DAD-300 detector, a Purospher
  • RP-18 end capped column a mobile phase of 15 % solvent A (0.1 % acetic acid in water) and 85 % solvent B (methanol) at a flow rate of 1.5 mL/min: Fl - retention time 0-5 minutes, F2 - retention time 5-9 minutes, F3 - retention time 9-12 minutes, F4 - retention time 12-14.5minutes, F5 - retention time 18-20, F6 - retention time 24-26, F7 - retention time 28-35 minutes, F8 - retention time 35-37, F9 - retention time 37-40
  • At least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more of the fractions may be pooled together, at any combination thereof.
  • the fraction comprises THCA.
  • THCA refers to A9-tetrahydrocannabinolic acid, the precursor of tetrahydrocannabinol (THC).
  • THC tetrahydrocannabinol
  • THCA as used herein encompasses native THCA (i.e. originating from the Cannabis plant), or synthetic analogs or derivatives thereof. Any THCA analog may be used in accordance with the present teachings as long as it comprises an antiinflammatory activity (alone, or as part of the composition discussed herein).
  • analog refers to a structural derivative having at least the same antiinflammatory activity.
  • the analog may be synthetic or naturally occurring.
  • Exemplary THCA analogs include, but are not limited to, l l-OH-delta9-THCA-A and 11-
  • Nor-delta9-THCA-A carboxylic acid [as discussed in detail in Guillermo Moreno-Sanz, Critical Review and Novel Therapeutic Perspectives of D9 -Tetrahydrocannabinolic Acid A, Cannabis and Cannabinoid Research Volume 1.1, (2016)].
  • the extracts and/or fractions comprise at least about 75-95 % THCA, at least about 80-90 % THCA, at least about 80-95 % THCA, at least about 80-100 % THCA, or at least about 90-100 % THCA.
  • the extracts and/or fractions comprise at least about 60 % THCA, at least about 65 % THCA, at least about 70 % THCA, at least about 75 % THCA, at least about 80 % THCA, at least about 81 % THCA, at least about 82 % THCA, at least about 83 % THCA, at least about 84 % THCA, at least about 85 % THCA, at least about 86 % THCA, at least about 87 % THCA, at least about 88 % THCA, at least about 89 % THCA, at least about 90 % THCA, at least about 91 % THCA, at least about 92 % THCA, at least about 93 % THCA, at least about 94 % THCA, at least about 95 % THCA, at least about 96 % THCA, at least about 97 % THCA, at least about
  • the cannabis extracts or fractions comprise 75 % or more THCA.
  • the cannabis extracts or fractions comprise 80 % or more THCA.
  • the cannabis extracts or fractions comprise 85 % or more THCA.
  • the cannabis extracts or fractions comprise 95 % or more THCA.
  • the extracts and/or fractions comprise cannabis derived active ingredients other than the THCA.
  • the extracts and/or fractions comprise at least one of D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), ⁇ 9 -tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the extracts and/or fractions comprise at least two, at least three, at least four, at least five, at least six, at least seven of D- Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), ⁇ 9- tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the extracts and/or fractions comprise D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and cannabigerol (CBG).
  • the extracts and/or fractions comprise THCA and any one, two, three, four, five, six or seven of D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), ⁇ 9 -tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the extracts and/or fractions comprise THCA, D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and cannabigerol (CBG).
  • Caryophyllene, Humulene, malic acid, ⁇ -Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and/or cannabigerol (CBG) may be provided as a synthetic analog.
  • the extracts and/or fractions comprise components as listed in Table 6, herein below.
  • the extracts and/or fractions of some embodiments of the invention comprise at least one, two, three, four, five, six, seven, eight, nine, ten or more components as listed in Table 6.
  • the extracts and/or fractions of some embodiments of the invention comprise THCA as well as at least one, two, three, four, five, six, seven, eight, nine, ten or more components as listed in Table 6.
  • the present invention is also directed to a product prepared by the process of the present invention.
  • an anti-inflammatory composition obtainable by the method of some embodiments of the invention.
  • a composition comprising liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, the active ingredients comprising THCA (at a level such as described hereinabove).
  • a composition comprising liquid chromatography-purified cannabis fraction obtainable by subjecting the cannabis extract to liquid chromatography and collecting fractions detectable by a detector operated at 220 nm.
  • the composition is characterized by: (i) having a cytotoxic activity on cancer cells (ii) reducing the level of pro-inflammatory cytokine secretion by cells; and/or (iii) reducing the level of MMP9 and COX2 expression in cells (e.g. inflammatory cells).
  • compositions comprising liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, the composition being characterized by: (i) having a cytotoxic activity on cancer cells; (ii) reducing the level of pro-inflammatory cytokine secretion by cells; and/or (iii) reducing the level of MMP9 and COX2 expression in cells (e.g. inflammatory cells).
  • the composition comprises THCA (e.g. as an active ingredient).
  • the composition comprises at least about 75-95 % THCA, at least about 80-90 % THCA, at least about 80-95 % THCA, at least about 80-100 % THCA, or at least about 90-100 % THCA.
  • the composition comprises at least about 60 % THCA, at least about 65 % THCA, at least about 70 % THCA, at least about 75 % THCA, at least about 80 % THCA, at least about 81 % THCA, at least about 82 % THCA, at least about 83 % THCA, at least about 84 % THCA, at least about 85 % THCA, at least about 86 % THCA, at least about 87 % THCA, at least about 88 % THCA, at least about 89 % THCA, at least about 90 % THCA, at least about 91 % THCA, at least about 92 % THCA, at least about 93 % THCA, at least about 94 % THCA, at least about 95 % THCA, at least about 96 % THCA, at least about 97 % THCA, at least about 98 % TH
  • the composition comprises 75 % or more THCA.
  • the composition comprises 80 % or more THCA.
  • the composition comprises 85 % or more THCA. According to a specific embodiment, the composition comprises 95 % or more THCA.
  • compositions may be supplemented with THCA (e.g. from pooled fractions, from THCA synthetic analogs, as discussed above).
  • the composition comprising THCA may comprise a dose range of THCA of 0.1-1000 mg/ml, 0.1-100 mg/ml, 0.1-50 mg/ml, 0.1-10 mg/ml, 0.1-5 mg/ml, 0.1-2.5 mg/ml, 0.1-1 mg/ml, 0.2-2000 mg/ml, 0.2-200 mg/ml, 0.2-20 mg/ml, 0.2-2 mg/ml, 1-1000 mg/ml, 10-100 mg/ml, 10-50 mg/ml, 2-2000 mg/ml, 20-200 mg/ml, 20-100 mg/ml, e.g. 20-30 mg/ml or e.g. 0.2-0.7 mg/ml.
  • the composition comprises THCA at a range of 5-100 mg/ml/gr of fresh cannabis, e.g. 20-30 mg/ml mg/ml/gr of fresh cannabis.
  • the composition comprises cannabis derived active ingredients other than the THCA.
  • the composition comprises at least one of D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), ⁇ 9- tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the composition comprises at least two, at least three, at least four, at least five, at least six, at least seven of D-Limonene, ⁇ - Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the composition comprises D- Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), ⁇ 9- tetrahydrocannabinol (THC), and cannabigerol (CBG).
  • the composition comprises THCA and any one, two, three, four, five, six or seven of D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the composition comprises THCA, D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and cannabigerol (CBG).
  • composition comprises components as listed in Table 6, herein below.
  • composition of some embodiments of the invention comprises at least one, two, three, four, five, six, seven, eight, nine, ten or more components as listed in Table 6.
  • compositions of some embodiments of the invention comprise THCA as well as at least one, two, three, four, five, six, seven, eight, nine, ten or more components as listed in Table 6.
  • the collective amount of the components in the fraction or composition does not exceed about 30 %, about 20 %, about 10 % or about 5 %.
  • the composition comprises cannabis derived active ingredients other than the THCA.
  • the extracts and/or fractions obtained by the methods of some embodiments of the invention may supplemented with components not present in the extract/fractions or present in the extract/fractions (such as to increase the level of a specific component).
  • the composition comprises CBD.
  • CBD cannabidiol
  • the term “CBD” refers to cannabidiol.
  • CBD as used herein encompasses native CBD (i.e. originating from the Cannabis plant), or synthetic analogs or derivatives thereof. Any CBD analog may be used in accordance with the present teachings as long as it comprises an anti-inflammatory activity (alone, or as part of the composition discussed herein).
  • Exemplary CBD analogs include, but are not limited to, (-)-DMH-CBD-l l-oic acid, HU- 308 (commercially available e.g. from Tocris Bioscience, 3088), O-1602 (commercially available e.g. from Tocris Bioscience 2797/10), DMH-CBD (commercially available e.g. from Tocris Bioscience, 1481) [as discussed in detail in Burstein S, Bioorg Med Chem. (2015) 23(7): 1377-85], Abn-CBD, HUF-101.
  • CBDV CBDM, CBND-C5, CBND-C3, 6-Hydroxy-CBD-triacetate or CBD- aldehyde-diacetate [as discussed in detail in An Overview on Medicinal Chemistry of Synthetic and Natural Derivatives of Cannabidiol, Frontiers in Pharmacology, June 2017 I Volume 8 I Article 422].
  • composition comprising THCA and CBD, wherein the composition is devoid of cannabichromene (CBC).
  • CBC cannabichromene
  • composition comprising THCA and CBD, wherein the composition is devoid of at least one of cannabichromene (CBC), cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), cannabigerol (CBG) and/or cannabinol (CBN).
  • CBC cannabichromene
  • CBD cannabigerolic acid
  • CBDA cannabidiolic acid
  • CBD cannabigerol
  • CBN cannabinol
  • composition comprising THCA and CBD, wherein the composition is devoid of cannabichromene (CBC), cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), cannabigerol (CBG) and cannabinol (CBN).
  • CBC cannabichromene
  • CBD cannabigerolic acid
  • CBDA cannabidiolic acid
  • CBD cannabigerol
  • CBN cannabinol
  • the composition comprises THCA, CBD and any one, two, three, four, five, six or seven of D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), ⁇ 9 -tetrahydrocannabinol (THC), and/or cannabigerol (CBG).
  • the composition comprises THCA, CBD, D-Limonene, ⁇ -Caryophyllene, Humulene, malic acid, a-Farnesene, cannabinol (CBN), A9-tetrahydrocannabinol (THC), and cannabigerol (CBG).
  • compositions of some embodiments of the invention comprise THCA, CBD as well as at least one, two, three, four, five, six, seven, eight, nine, ten or more components as listed in Table 6.
  • the different components e.g. THCA and CBD
  • the different components are discrete i.e. not comprised in the same fraction.
  • the fractions are combined into a single composition.
  • the fractions are comprised in different formulations/compositions .
  • composition comprising THCA and CBD may comprise a dose range of THCA as discussed above.
  • composition comprising
  • THCA and CBD may comprise a dose range of CBD of 0.01-1000 mM, 0.01-100 mM, 0.01-10 mM, 0.01-1 mM, 0.1-1000 mM, 0.1-500 mM, 0.1-100 mM, 0.1-50 mM, 0.1-10 mM, 0.1-5 mM, 0.1-2.5 mM/, 0.1-1 mM, 0.5-50 mM, 0.5-10 mM, 0.5-5 mM, 0.5-1 mM, e.g. 0.22-0.45 mM or 0.01-0.03.
  • extracts of the present invention active fractions derived therefrom, and compositions comprising same have anti-inflammatory activity, they may be used for treating diseases or disorders related thereto.
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of some embodiments of the invention, thereby treating the inflammatory disease in the subject.
  • composition of some embodiments of the invention for use in treating an inflammatory disease in a subject in need thereof.
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a liquid chromatography fraction of a cannabis extract comprising at least about 75 % tetrahydrocannabinolic acid (THCA), wherein the fraction comprises cannabis derived active ingredients other than the THCA, thereby treating the inflammatory disease in the subject.
  • THCA tetrahydrocannabinolic acid
  • a therapeutically effective amount of a liquid chromatography fraction of a cannabis extract comprising at least about 75 % tetrahydrocannabinolic acid (THCA), wherein the fraction comprises cannabis derived active ingredients other than the THCA, for use in treating an inflammatory disease in a subject in need thereof.
  • THCA tetrahydrocannabinolic acid
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a composition of matter comprising a therapeutically effective amount of liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, wherein the active ingredients comprise THCA, thereby treating the inflammatory disease in the subject.
  • a therapeutically effective amount of liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, wherein the active ingredients comprise THCA, for use in treating an inflammatory disease in a subject in need thereof.
  • THCA has anti-inflammatory activity on its own, it is proposed that THCA can be used as is for the treatment of inflammatory disease in a subject in need thereof.
  • a method of treating an inflammatory disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising tetrahydrocannabinolic acid (THCA), wherein the THCA constitutes at least about 30 % of the active ingredients in the composition, thereby treating the inflammatory disease in the subject.
  • THCA tetrahydrocannabinolic acid
  • THCA tetrahydrocannabinolic acid
  • the THCA constitutes at least about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 75 %, about 80 %, about 85 %, about 90 %, about 95 %, about 99 %, or about 100 % of the active ingredients in the composition.
  • the term "subject” or “subject in need thereof” refers to a mammalian e.g., human subject, at any age or gender, who has an inflammatory disease.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Inflammatory diseases include chronic inflammatory diseases and acute inflammatory diseases.
  • hypersensitivity examples include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.
  • Type I or immediate hypersensitivity such as asthma.
  • Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul; 15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol.
  • vasculitises necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178); antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999; 14 (4): 171); heart failure, agonist-like ⁇ -adrenoceptor antibodies in heart failure (Wallukat G.
  • Type IV or T cell mediated hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18;91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol.
  • delayed type hypersensitivity examples include, but are not limited to, contact dermatitis and drug eruption.
  • T lymphocyte mediating hypersensitivity examples include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.
  • helper T lymphocyte-mediated hypersensitivity examples include, but are not limited to, T h l lymphocyte mediated hypersensitivity and T h 2 lymphocyte mediated hypersensitivity.
  • Autoimmune diseases include, but are not limited to, T h l lymphocyte mediated hypersensitivity and T h 2 lymphocyte mediated hypersensitivity.
  • cardiovascular diseases include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
  • autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al., Lupus. 1998;7 Suppl 2:S 135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S 132), thrombosis (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al., Wien Klin Klin Klin Klinschr 2000 Aug 25;112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix- Desmazes S.
  • autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189).
  • autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome.
  • Diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S 125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J.
  • autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16;138 (2): 122), colitis, ileitis and Crohn's disease.
  • autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
  • autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999 Jun;l l (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).
  • autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer's disease (Oron L. et al, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci.
  • autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et ah, Int Arch Allergy Immunol 2000 Sep; 123 (1):92) and smooth muscle autoimmune disease (Zauli D. et ah, Biomed Pharmacother 1999 Jun;53 (5-6):234).
  • autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; l (2): 140).
  • autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et ah, Lupus 1998;7 Suppl 2:S 107-9).
  • autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et ah , Cell Immunol 1994 Aug; 157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et ah , Ann N Y Acad Sci 1997 Dec 29;830:266).
  • autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et ah, Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et ah, Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et ah, Immunol Rev 1999 Jun; 169: 107).
  • infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.
  • diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.
  • allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.
  • the inflammatory disease is an inflammatory bowel disease (IBDs).
  • IBDs inflammatory bowel disease
  • CD Crohn's disease
  • UC ulcerative colitis
  • the method further comprises administering to the subject a therapeutically effective amount of cannabidiol (CBD).
  • CBD cannabidiol
  • the fraction, fractions, THCA or composition for use further comprises the use of a therapeutically effective amount of cannabidiol (CBD).
  • CBD may be administered prior to, concomitantly with, or following administration of the composition of some embodiments of the invention.
  • the method further comprises administering to the subject an agonist of CB l receptor, CB2 receptor and/or GPR55.
  • the fraction, fractions, THCA or composition for use further comprises the use of an agonist of CB l receptor, CB2 receptor and/or GPR55.
  • an agonist of CB l receptor comprises, for example, nabilone, WIN 55,212-2, HU210, and anandamide.
  • an agonist of CB2 receptor comprises, for example, AM1241, GW405833, JWH133 and WIN 55,212-2.
  • an agonist of GPR55 receptor comprises, for example, 2-arachidonoylglycerolphosphoinositol.
  • the agonist of CB l receptor, CB2 receptor and/or GPR55 may be administered prior to, concomitantly with, or following administration of the composition of some embodiments of the invention.
  • compositions described hereinabove can be administered to the individual per se or as part of a pharmaceutical composition which also includes physiologically acceptable carriers or excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the cannabis derived active ingredients accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • tissue refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
  • Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the composition can be formulated in a form of a gel, a cream, an ointment, a paste, a lotion, a milk, a suspension, an aerosol, a spray, a foam, a serum, a swab, a pledget, a pad or a patch.
  • Formulations for transdermal delivery can typically include carriers such as water, liquid alcohols, liquid glycols, liquid polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates, liquid alkylated protein hydrolysates, liquid lanolin, lanolin derivatives, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and like materials commonly employed in topical compositions.
  • Various additives may be included in the transdermal formulations of the invention. For example, solvents may be used to solubilize certain active ingredients substances.
  • Other optional additives include skin permeation enhancers, opacifiers, anti-oxidants, gelling agents, thickening agents, stabilizers, and the like.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (cannabis derived active ingredients) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., IBD) or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • a therapeutically effective amount of a composition comprising liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, wherein the active ingredients comprise THCA, is in the range of 0.1-1000 mg/day/kg, 0.1-100 mg/day/kg, 0.1-50 mg/day/kg, 0.1-10 mg/day/kg, 0.1-5 mg/day/kg, 0.1-2.5 mg/day/kg, 0.1-1 mg/day/kg, 0.2-1.5 mg/day/kg, e.g. 0.2-0.7 mg/day/kg, e.g. 0.7 mg/day/kg.
  • a therapeutically effective amount of a composition comprising a therapeutically effective amount of a liquid chromatography fraction of a cannabis extract comprising at least about 30 % THCA (e.g. 60-100 % THCA e.g. 75 % THCA), wherein the fraction comprises cannabis derived active ingredients other than the THCA, is in the range of 0.1- 1000 mg/day/kg, 0.1-500 mg/day/kg, 0.1-100 mg/day/kg, 0.1-50 mg/day/kg, 0.1-10 mg/day/kg, 0.1-5 mg/day/kg, 0.1-2.5 mg/day/kg, 0.1-1 mg/day/kg, 0.2-1.5 mg/day/kg, e.g. 0.2-0.7 mg/day/kg, e.g. 0.7 mg/day/kg.
  • CBD when CBD is administered in conjunction with a composition comprising liquid chromatography pooled fractions of cannabis extract comprising active ingredients detectable by a detector operated at 220 nm, wherein the active ingredients comprise THCA, a therapeutically effective amount is based on the relation of the CBD to the composition.
  • CBD when administered in conjunction with a composition comprising a therapeutically effective amount of a liquid chromatography fraction of a cannabis extract comprising at least about 30 % THCA (e.g. 60-100 % THCA e.g. 75 % THCA), wherein the fraction comprises cannabis derived active ingredients other than the THCA, a therapeutically effective amount is based on the relation of the CBD to the composition.
  • a composition comprising a therapeutically effective amount of a liquid chromatography fraction of a cannabis extract comprising at least about 30 % THCA (e.g. 60-100 % THCA e.g. 75 % THCA), wherein the fraction comprises cannabis derived active ingredients other than the THCA
  • a therapeutically effective amount is based on the relation of the CBD to the composition.
  • CBD can be administered at a dose range of 0.1-1000 mM/day/kg, 0.1-500 mM/day/kg, 0.1-100 mM/day/kg, 0.1-50 mM/day/kg, 0.1-10 mM/day/kg, 0.1- 5 mM/day/kg, 0.1-2.5 mM/day/kg, 0.1-1 mM/day/kg, 0.5-50 mM/day/kg, 0.5-10 mM/day/kg, 0.5-5 mM/day/kg, 0.5-1 mM/day/kg, e.g. 0.22-0.45 mM/day/kg.
  • composition when the composition is administered at a dose of 0.7 mg/day/kg (wherein the active ingredients comprise THCA), CBD is administered at a dose of 0.45 mM/day/kg, i.e. at a relation of 1.5: 1 (composition:CBD).
  • composition:CBD when the composition is administered at a dose of 0.7 mg/day/kg (wherein the active ingredients comprise THCA), CBD is administered at a dose of 0.22 mM/day/kg, i.e. at a relation of 3: 1 (composition:CBD).
  • composition:CBD composition:CBD
  • CBD when CBD is administered at a dose of 0.4 mM/day/kg, the composition (wherein the active ingredients comprise THCA) is administered at a dose of 0.4 mg/day/kg, i.e. at a relation of 1: 1 (CBD omposition).
  • CBD when CBD is administered at a dose of 0.4 mM/day/kg, the composition (wherein the active ingredients comprise THCA) is administered at a dose of 0.3 mg/day/kg, i.e. at a relation of 1.33: 1 (CBD omposition).
  • the composition when CBD is administered at a dose of 0.4 mM/day/kg, the composition (wherein the active ingredients comprise THCA) is administered at a dose of 0.2 mg/day/kg, i.e. at a relation of 2: 1 (CBD omposition).
  • CBD omposition a relation of 2: 1
  • Such a dosing can be adjusted as long as the relation of composition:CBD or CBD: composition is maintained as described above, a person of skill in the art can make the proper adjustments based on the subject being treated and the level of the components (e.g. THCA) in the composition.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).
  • Dosage amount and interval may be adjusted individually to provide cannabis derived active ingredients (the intestinal tissue) levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the present invention in order to enhance treatment of the inflammatory disease, further envisions administering to the subject an additional therapy which may benefit treatment.
  • an additional therapy which may benefit treatment.
  • One of skill in the art is capable of making such a determination.
  • the anti-inflammatory therapy may include, without being limited to, NSAIDs (Non- Steroidal Anti-inflammatory Drugs), corticosteroids (such as prednisone) and antihistamines.
  • NSAIDs Non- Steroidal Anti-inflammatory Drugs
  • corticosteroids such as prednisone
  • antihistamines antihistamines
  • Additional anti-inflammatory agents which may be used according to the present teachings include, but are not limited to, Alclofenac; Alclometasone Dipropionate; Algestone Acetonide;
  • Anirolac Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine
  • Enlimomab Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen;
  • Fenclofenac Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl;
  • Fluorometholone Acetate Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;
  • Furaprofen Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac;
  • Ibuprofen Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin
  • Meclofenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Meclorisone Dibutyrate
  • Mefenamic Acid Mefenamic Acid
  • Mesalamine Meclorisone Dibutyrate
  • Meseclazone
  • Salcolex Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam;
  • Sulindac Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; Zomepirac Sodium.
  • Any of the above described agents may be administered individually or in combination.
  • the cannabis extract of the present invention can be administered to a subject (e.g., a human) in need thereof in a variety of other forms including a nutraceutical composition.
  • a "nutraceutical composition” refers to any substance that may be considered a food or part of a food and provides medical or health benefits, including the prevention and treatment of disease.
  • a nutraceutical composition is intended to supplement the diet and contains at least one or more of the following ingredients: a vitamin; a mineral; an herb; a botanical; a fruit; a vegetable; an amino acid; or a concentrate, metabolite, constituent, or extract of any of the previously mentioned ingredients; and combinations thereof.
  • a nutraceutical composition of the present invention can be administered as a "dietary supplement," as defined by the U.S. Food and Drug Administration, which is a product taken by mouth that contains a "dietary ingredient” such as, but not limited to, a vitamin, a mineral, an herb or other botanical, an amino acid, and substances such as an enzyme, an organ tissue, a glandular, a metabolite, or an extract or concentrate thereof.
  • a dietary supplement as defined by the U.S. Food and Drug Administration, which is a product taken by mouth that contains a "dietary ingredient” such as, but not limited to, a vitamin, a mineral, an herb or other botanical, an amino acid, and substances such as an enzyme, an organ tissue, a glandular, a metabolite, or an extract or concentrate thereof.
  • Non-limiting forms of nutraceutical compositions of the present invention include: a tablet, a capsule, a pill, a softgel, a gelcap, a liquid, a powder, a solution, a tincture, a suspension, a syrup, or other forms known to persons of skill in the art.
  • a nutraceutical composition can also be in the form of a food, such as, but not limited to, a food bar, a beverage, a food gel, a food additive/supplement, a powder, a syrup, and combinations thereof.
  • a method of determining an anti-inflammatory activity of the composition of some embodiments of the invention comprising ex-vivo contacting an inflamed tissue of a subject with the composition, wherein an increased anti-inflammatory response of the inflamed tissue above a predetermined threshold is indicative of the anti-inflammatory activity of the composition.
  • the inflamed tissue is a gastrointestinal tissue biopsy.
  • Exemplary tissues include oesophagus, gallbladder, liver, pancreas, stomach, small intestine, bowel (large intestine or colon and rectum), and anus.
  • the inflamed tissue is obtained from a subject at an active stage of an inflammatory disease.
  • inflammatory diseases include, but are not limited to, IBD, UC and Crohn's disease.
  • the anti-inflammatory activity of the composition comprises an upregulation in secretion of an anti-inflammatory factor (e.g. cytokine such as, but not limited to, IL-4, IL-10, IL-13, IFN-alpha and TGF-beta) and/or reduction in secretion of a proinflammatory factor (e.g. cytokine such as, but not limited to, IL-6, IL-8, IL- ⁇ , TNF-a, INF- ⁇ , IL- 12, IL-18 and GM-CSF).
  • an anti-inflammatory factor e.g. cytokine such as, but not limited to, IL-4, IL-10, IL-13, IFN-alpha and TGF-beta
  • a proinflammatory factor e.g. cytokine such as, but not limited to, IL-6, IL-8, IL- ⁇ , TNF-a, INF- ⁇ , IL- 12, IL-18 and GM-CSF.
  • the anti-inflammatory activity of the composition comprises reduction in expression of a gene associated with the inflammation.
  • exemplary genes include, but are not limited to, MMP9 and COX2.
  • a predetermined threshold can be established by determining an expression level (e.g. of a gene) or secretion of a factor (e.g. cytokine) by a healthy tissue (e.g. of a healthy donor subject, of the subject before disease onset or during disease remission, or from tissue cultures available commercially).
  • an expression level e.g. of a gene
  • a factor e.g. cytokine
  • a healthy tissue e.g. of a healthy donor subject, of the subject before disease onset or during disease remission, or from tissue cultures available commercially.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • Fresh flowers of C. sativa strain AD were harvested from plants. They were either taken immediately for extraction and frozen at -80 °C, or baked for 3 hours at 150 °C prior to extraction.
  • Fresh and baked Cannabis flowers (2 g) were pulverized with liquid nitrogen. Absolute ethanol was added to each tube containing the powder at a sample-to-absolute ethanol ratio of 1:4 (w/v). The tubes were mixed thoroughly on a shaker for 30 minutes and then the extract was filtered through a filter paper. The filtrate was transferred to new tubes. The solvent was evaporated with a vacuum evaporator. The dried extract was resuspended in 1 mL of absolute methanol and filtered through a 0.45- ⁇ syringe filter.
  • the filtered liquid was collected for the treatments, the resuspended extract was diluted for cell cultures and biopsies in enzyme-linked immunosorbent assay (ELISA) experiments.
  • Sample dry weight was determined by crushing 1 g of plant material with known fresh weight and incubating overnight at 60 °C, then weighing again for dry weight calculation.
  • the cannabinoid standards cannabigerol (CBG), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabigerolic acid (CBGA), tetrahydrocannabinol (THC), cannabichromene (CBC) and tetrahydrocannabinolic acid (THCA) were diluted to 10 ppm concentration with methanol and then subjected to HPLC separation.
  • the standards were dissolved in methanol at different concentrations from 5 ppm to 40 ppm.
  • the dry extract (the ethanol crude) was resuspended in 1 mL methanol and filtered through a 0.45- ⁇ syringe filter (Merck, Darmstadt, Germany).
  • the filtered extract (the filtrate) was diluted 10 times with methanol and then separated by HPLC. For profile, the filtrate was diluted 50 times with methanol.
  • GC/MS analyses were carried out using a HP7890 gas chromatograph coupled to a HP6973 mass spectrometer with electron multiplier potential 2 KV, filament current 0.35 mA, electron energy 70 eV, and the spectra were recorded over the range m/z 40 to 400.
  • An Agilent 7683 autosampler was used for sample introduction. Helium was used as a carrier gas at a constant flow of 1.1 ml s-1.
  • One ⁇ of each sample was injected to the GC/MS using a 1: 10 split ratio injection mode.
  • An isothermal hold at 50 °C was kept for 2 minutes, followed by a heating gradient of 6 °C min-1 to 300 °C, with the final temperature held for 4 minutes.
  • HCT116 ATCC CCL-247
  • HT29 ATCC HTB-38
  • CaC02 ATCC HTB-37 colon cells were grown at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere.
  • Cells were maintained in McCoy's 5a Modified Medium (HCT116 and HT29) and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (CaC02).
  • DMEM Dulbecco's modified Eagle's medium
  • IL-8 [12] was measured 4-16 hours post-treatment using the commercial Human CXCL8/IL-8 DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA).
  • dexamethasone Sigma-Aldrich, St. Louis, MO, USA
  • 200 and 400 ⁇ final concentration was used.
  • CB 1, CB2 and GPR55 The involvement of the receptors (CB 1, CB2 and GPR55) was examined by treating the cells with 20 ⁇ of the CB 1 receptor antagonist/inverse agonist Rimonabant (Abeam, Cambridge, MA, USA), CB2 receptor antagonist/inverse agonist SR144528 (Abeam) and GPR55 antagonist/inverse agonist CID 16020046 (Sigma-Aldrich, Buchs, Switzerland).
  • the whole extract from fresh flowers (C2F) or the active fraction (F7) was applied to cells along with TNF-a 1 hour after the antagonist treatment.
  • biopsies from both healthy and inflamed intestine of IBD (inflammatory bowel disease) patients were obtained from 29 patients with either Crohn's disease (CD) or ulcerative colitis (UC) scheduled for colonoscopy as deemed necessary by their physician, Helsinki approval no. 0094- 16 was obtained.
  • biopsies from inflamed and normal tissue were taken and placed in tissue culture media.
  • PBS was replaced with 75 ⁇ ⁇ dispase (StemCell Technologies, Cambridge, UK) and 150 ⁇ ⁇ collagenase 1A (StemCell Technologies) solution. Tubes were then incubated at 37 °C for 1 hour.
  • the tubes containing the biopsies were centrifuged at 8000 rpm (11,885 x g) for 1 minute. Then the supernatant was removed and tissues were washed three times with Hank's balanced salt solution. After each wash, tubes were centrifuged as described above. Then the tissues were placed on a small petri dish and cut into 2-3 pieces with a clean scalpel. The pieces were then placed on Millicel hydrophilic PTFE tissue-culture inserts (Millipore, 30 mm, 0.4 ⁇ ).
  • tissue-culture dishes Costar 3506 along with 1.5 mL of tissue-culture medium (Dulbecco' s modified Eagle' s medium supplemented with 10 % v/v heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 50 ⁇ g/mL leupeptin, 1 mM PMSF, and 50 ⁇ g/mL soybean trypsin inhibitor).
  • tissue-culture medium Dulbecco' s modified Eagle' s medium supplemented with 10 % v/v heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 50 ⁇ g/mL leupeptin, 1 mM PMSF, and 50 ⁇ g/mL soybean trypsin inhibitor.
  • dexamethasone was included in the media, its concentration was 200 ⁇ g/mL. This was followed by treating the tissues with extracts as mentioned above, or leaving them untreated
  • TNF-a When TNF-a was added to induce interleukins (IL) expression, the concentration was 50 ng/mL. Cultures were then incubated at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere. To evaluate TNF-a expression, supernatants were taken after 1 hour of incubation. For other ILs, supernatants were taken after overnight incubation unless otherwise stated. The supernatants from the biopsies were used for determination of IL-8 cytokine profile by measuring its levels with a commercial ELISA kit. Levels of cytokines from inflamed, cannabis-treated and non-treated tissue were compared.
  • Cells were seeded into a 6-well plate at a concentration of 1,500,000 cell/mL per well. After 24 hours incubation at 37 °C in a humidified 5 % C0 2 -95 % air atmosphere, cells were treated with TNF-a (final concentration of 1 ng/mL) and incubated overnight under the same conditions. Non- treated cells or cells treated only with TNF-a served as negative and positive controls, respectively. Cells were then reincubated for 5 hours with C2F (0.2 mg crude dry extract/mL) or F7 (0.08 mg/mL) at 37°C in a humidified 5% C0 2 -95% air atmosphere.
  • C2F 0.2 mg crude dry extract/mL
  • F7 0.08 mg/mL
  • the primers were: for COX2 (forward, SEQ ID NO: 1) 5 ' - ATTGACC AGAGC AGGC AGAT-3 ' and (reverse, SEQ ID NO: 2) ' -CAGG ATAC AGCTCCAC AGC A-3 ' , and for MMP9 (forward, SEQ ID NO: 3) 5 * - TTGACAGCGACAAGAAGTGG-3' and (reverse, SEQ ID NO: 4) 5'- TC AC GTC GTCCTT ATGC A AG- 3 ' .
  • ELISA assay was used to measure the anti-inflammatory activity of the fractions on HCT 1 16 cells.
  • Cells were seeded into a 24-well plates at 50,000 cells per well in triplicate in normal growing media. After 24 hours of incubation at 37 °C in a humidified 5 % C02-95 % air atmosphere, cells were treated with different concentration of F7 (5 ⁇ g/mL to 80 ⁇ g/mL) with and without CBD (30 ⁇ ) along with 300 ng/niL of TNF-a or with different concentration of CBD (5 ⁇ to 120 ⁇ ) with and without F7 (30 ⁇ g/mL) along with 300 ng/niL of TNF-a for 4 hours.
  • (Exy) is the additive effect of the drug x and y as predicted by their individual effects (Ex and Ey).
  • the drug's anti- cancer effect was defined as complementary to the obtained results (1-Exy).
  • the observed combined percentage viability is then compared to the calculated value. If the observed value of Exy is greater than the calculated Exy value, the combination treatment is considered as worse than expected, which means antagonism effect. If the observed value is less than the calculated one, then the combination treatment is considered as better than expected, thus showing synergism effect. If both values are equal, the combination treatment is considered as the same for the addition of the two drugs, which means additive effect (independent).
  • Results are presented as mean + SE of replicate analyses and are either representative of or include at least two independent experiments. Means of replicates were subjected to statistical analysis by Tukey-Kramer test (P ⁇ 0.05) using the JMP statistical package and considered significant when P ⁇ 0.05.
  • C. sativa extracts from fresh flowers are highly active in reducing inflammation in colon cell lines
  • Anti-inflammation activity was determined for absolute ethanol extracts of fresh (C2F) and baked (C2B) flowers of C. sativa (Cs-AD var.). The activity was determined as the level of reduction of IL-8 in HCT116 colon cancer cell cultures pretreated with TNF-a to induce IL-8 expression and then treated with C2F or C2B ( Figure IB and IE). Notably, IL-8 was used in several other studies, in HCT116 as well as other cell models and in IBD patients as an indicator for the level of IBD-related inflammation [Ihenetu K. et al., Eur J Pharmacol. (2003) 458:207-215; Banks C et al., J Pathol. (2003) 199:28-35].
  • cannabinoids were identified in the fresh and baked crude extracts at 220 nm. These peaks were identified as CBG, CBD, CBDA, CBN, CBGA, THC, CBC and THCA, with retention times of 5.9, 6.4, 7.9, 10.9, 11.3, 13.1, 17.5 and 29.3 minutes, respectively, relative to the HPLC profile of cannabinoid standards (not shown).
  • the levels of CBD, CBGA and THC were 36, 14 and 32 times higher in the C2B versus C2F extract. CBC was not identified in C2F but appeared in C2B.
  • C2F (at a concentration of 163 ⁇ g/mL) was fractionated (Figure 3 A) by HPLC. Fractions were collected and high concentrations (0.9 mg/ml) were examined for anti-inflammatory activity, determined as the level of IL-8 in HCTl 16 cells.
  • CBD reduces inflammation only at lower doses yet its cytotoxic activity is dose dependent
  • CBD was active in reduction of IL-8 levels in CaC02 and HT29 cells (Figure 31). However, treatments with CBD lead to a dose-dependent cell death in HCT116 and in CaC02 cells, and to a lesser extent in HT29 cells ( Figure 3 J and Figure 6).
  • the active fraction of C. sativa extract contains mainly THCA
  • F7 The chemical composition of the active fraction (F7) was analyzed by HPLC and electrospray ionization mass spectrometry (ESI-MS). F7 was obtained as a broad peak in the HPLC chromatogram. To analyze its structure and verify its purity, it was analyzed at different dilutions, in comparison to a THCA standard. The results suggested that F7 is THCA ( Figure 4). ESI-MS results further confirmed that F7 contains THCA: C 2 2H 30 O 4 (358.214); m z (MH+) 359.222, (MNa+) 381.203. 1H and 13 C spectra were taken to verify the exact structure and determine the purity of F7.
  • ESI-MS electrospray ionization mass spectrometry
  • THCA is active against inflammation in HCT116 cells
  • THCA was found to be the main compound in the active fraction of the C. sativa extract
  • the anti-inflammatory activity of the THCA (commercially available with purity of 99 %) in HCT116 cells was determined.
  • Treatments with THCA significantly reduced IL-8 levels in HCT116 cells (Figure 5).
  • To further determine whether the activity in F7 derives only from THCA its concentration in this fraction was determined to be 22.81 mg/niL, and F7 was diluted 280-fold, to correspond to 0.2 mM THCA. Under these conditions, the activities of F7 and THCA were similar (Figure 5), further suggesting that the antiinflammatory activity of F7 derives from that of THCA.
  • THCA but not CBD
  • the whole cannabis extract, C2F, or the pooled fractions have higher activity than that of F7 or F1-F9 excluding F7, evident also for relatively low concentrations of the fractions. This suggests interactions between fractions such that the combination of F7 (THCA) and compound(s) present in C. sativa fresh extract is more potent than F7 (THCA) only, in reducing HCT116 cell inflammation.
  • GPR55 receptor antagonist significantly reduces the anti-inflammatory activity ofF7, whereas
  • CB2 receptor antagonist significantly increases HCT116 cell proliferation
  • Transcripts for CB1, CB2 and GPR55 were detected by qPCR in HCT116 cells. Expression of CB2 and GPR55 were significantly increased upon treatment with TNF-a in these cells (values are the steady-state level of gene expression in TNF-a-treated versus non-treated cells; Table 2).
  • COX2 expression is induced in the large intestine of IBD patients (reviewed by [24]) and MMP9 is among the predominant proteinases expressed in the gut mucosa during active IBD, associated with disease severity [25].
  • MMP9 and COX2 expression were examined as markers for inflammation level in HCT116 cells and colon biopsies of IBD patients (four UC and one CD).
  • Expression of both COX2 and MMP9 was significantly induced in HCT116 cells treated with TNF-a and significantly reduced by treatment with C2F and F7.
  • F7 was more effective at reducing COX2 expression than C2F (Figure 9A).
  • FIG. 10 illustrates the dose response (in the anti-inflammatory activity) of F7 in HT29 cells after 4 hours of treatment. As evident from the results, treatment with F7 leads to a dose dependent reduction of IL- 8 levels in HT29 cells.
  • Figure 11 illustrates the dose response (in the anti-inflammatory activity) of CBD in HT29 cells after 4 hours of treatment.
  • FIG. 12 illustrates the dose response (in the anti-inflammatory activity) of THC in HT29 cells after 4 hours of treatment. As evident from the results, treatment with THC leads to a dose dependent reduction of IL-8 levels in HT29 cells.
  • the EC50 of F7, CBD and THC were calculated from the anti-inflammatory assay (50 % of activity) ( Figure 13).
  • the EC50 of F7, CBD and THC were determined to be 35.14, 30.35 and 102.2 ⁇ g/ml, respectively. These concentrations were further used in the next experiments of combinations of treatments.
  • CBD tetrahydrofuran
  • THCA tetrahydrofuran
  • fraction F7 contains THCA, THC, CBD and CBN, additional acids (palmitic acid, Linolenic acid, malic acid, Arachidonic acid, stearic acid and myristic acid) and compounds as detailed in Table 6, below

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Abstract

La présente invention a trait à une méthode de traitement d'une maladie inflammatoire chez un sujet qui en a besoin. La méthode comprend l'administration au sujet d'une quantité thérapeutiquement efficace d'une fraction de chromatographie liquide d'un extrait de cannabis comprenant au moins 75 % d'acide tétrahydrocannabinolique (THCA), la fraction comprenant des ingrédients actifs dérivés du cannabis autres que le THCA.
PCT/IL2018/050248 2017-03-05 2018-03-05 Compositions et méthodes pour le traitement de maladies inflammatoires Ceased WO2018163163A1 (fr)

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CA3055271A CA3055271A1 (fr) 2017-03-05 2018-03-05 Compositions et methodes pour le traitement de maladies inflammatoires
US16/491,197 US11833131B2 (en) 2017-03-05 2018-03-05 Compositions and methods for treating inflammatory diseases
CN201880026618.1A CN110799186A (zh) 2017-03-05 2018-03-05 治疗炎性疾病的组合物和方法
IL269158A IL269158B2 (en) 2017-03-05 2018-03-05 Cannabis preparations enriched with THCA and their use for the treatment of inflammatory diseases

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US11622956B1 (en) 2019-06-26 2023-04-11 RCR BioPharma Compound and method for treating diseases and disorders
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CN120303244A (zh) * 2022-12-02 2025-07-11 博迪贺康(绍兴)生物技术有限公司 一种化合物及其在Treg相关疾病的治疗中的应用

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IL269158B2 (en) 2024-04-01
US11833131B2 (en) 2023-12-05
CN110621312A (zh) 2019-12-27
EP3592346A1 (fr) 2020-01-15
EP3592345A4 (fr) 2020-12-30
IL269157A (en) 2019-11-28
US11331301B2 (en) 2022-05-17
IL269158B1 (en) 2023-12-01
US20200030282A1 (en) 2020-01-30
CN110799186A (zh) 2020-02-14
IL269158A (en) 2019-11-28
EP3592345A1 (fr) 2020-01-15
EP3592346A4 (fr) 2020-12-30

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